Thomas B. Knudsen

Modulation of TLR2 Protein Expression by miR-105 in Human Oral Keratinocytes

Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and regulate innate immune responses. Our model system comprised primary human keratinocytes, which exhibited robust differences in inflammatory cytokine production (interleukin-6 and tumor necrosis factor-α) following specific Toll-like receptor 2 and 4 (TLR-2/TLR-4) agonist challenge. We challenged these primary cells with Porphyromonas gingivalis (a Gram-negative bacterium that triggers TLR-2 and TLR-4) and performed miRNA expression profiling. We identified miRNA (miR)-105 as a modulator of TLR-2 protein translation in human gingival keratinocytes. There was a strong inverse correlation between cells that had high cytokine responses following TLR-2 agonist challenge and miR-105 levels. Knock-in and knock-down of miR-105 confirmed this inverse relationship. In silico analysis predicted that miR-105 had complementarity for TLR-2 mRNA, and the luciferase reporter assay verified this. Further understanding of the role of miRNA in host responses may elucidate disease susceptibility and suggest new anti-inflammatory therapeutics.

Reprogramming of genetic networks during initiation of the Fetal Alcohol Syndrome

Fetal Alcohol Spectrum Disorders (FASD) are birth defects that result from maternal alcohol use. We used a non a priori approach to prioritize candidate pathways during alcohol‐induced teratogenicity in early mouse embryos. Two C57BL/6 substrains (B6J, B6N) served as the basis for study. Dosing pregnant dams with alcohol (2× 2.9 g/kg ethanol spaced 4 hr on day 8) induced FASD in B6J at a higher incidence than B6N embryos. Counter‐exposure to PK11195 (4 mg/kg) significantly protected B6J embryos but slightly promoted FASD in B6N embryos. Microarray transcript profiling was performed on the embryonic headfold 3 hr after the first maternal alcohol injection (GEO data series accession GSE1074). This analysis revealed metabolic and cellular reprogramming that was substrain‐specific and/or PK11195‐dependent. Mapping ethanol‐responsive KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed down‐regulation of ribosomal proteins and proteasome, and up‐regulation of glycolysis and pentose phosphate pathway in B6N embryos; and significant up‐regulation of tight junction, focal adhesion, adherens junction, and regulation of the actin cytoskeleton (and near‐significant up‐regulation of Wnt signaling and apoptosis) pathways in both substrains. Expression networks constructed computationally from these altered genes identified entry points for EtOH at several hubs (MAPK1, ALDH3A2, CD14, PFKM, TNFRSF1A, RPS6, IGF1, EGFR, PTEN) and for PK11195 at AKT1. Our findings are consistent with the growing view that developmental exposure to alcohol alters common signaling pathways linking receptor activation to cytoskeletal reorganization. The programmatic shift in cell motility and metabolic capacity further implies cell signals and responses that are integrated by the mitochondrial recognition site for PK11195. Developmental Dynamics 236:613–631, 2007.

Embryotoxic, teratogenic, and metabolic effects of ribavirin in mice☆

Abstract To gain comparative perspective on the teratogenic mechanisms of action of drugs that are precursor analogs or antimetabolites of nucleic acids, we employed a new antiviral agent—ribavirin—which is known in other systems to inhibit the biosynthesis of guanine nucleotides. Pregnant ICR mice were injected at 10th–13th days of gestation (stages 14–20) with a single ip dose of ribavirin in the range of 10–200 mg/kg. All dosages in excess of 25 mg/kg were teratogenic. The optimal teratogenic dose varied with the stage of development, being higher at advanced stages of development. Depending on the dose and stage of treatment, virtually all parts of the skeleton including the craniofacial and limb bones were susceptible to ribavirin. Both the frequency and multiplicity of skeletal defects increased as the dose was raised. The stage dependency of defects in the orofacial bones was markedly apparent. Treatment on Day 10.5 resulted in shortened maxilla in all survivors, while treatment on either Day 11 or 11.5 resulted in a high frequency of reduction in the length of both upper and lowerjaws. Treatment on the 12th day resulted in a very low incidence of effect on the maxilla (4%) but a high frequency (88–100%) of reduction and deformation of the mandible. This enhanced susceptibility of individual facial bones at different stages of development with virtually no overlap provides an experimental model to study cellular phenomena underlying normal and abnormal facial development. Ribavirin, both in vivo and in vitro , inhibited embryonic DNA synthesis. The inhibition was transitory and did not seem to be directly related to the embryolethal activity of the drug. Although the role of metabolic inhibition in precipitating teratogenesis is not clear, cytotoxic action of ribavirin against proliferating limb bud mesenchymal cells is directly associated with the origin of limb deformities.

Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399Ile are hypo-responsive to Porphyromonas gingivalis

The Toll-like receptor (TLR)4 is the major sensor for bacterial lipopolysaccharide and its two common co-segregating polymorphisms, Asp299Gly and Thr399Ile, which occur at a frequency of between 6 and 10%, have been associated with infectious diseases, LPS hypo-responsiveness and cardiovascular disease. Porphyromonas gingivalis is a Gram-negative bacterium implicated in chronic periodontitis and is a known TLR4 and TLR2 agonist. We obtained two gingival epithelial cell primary cultures from subjects heterozygous for the TLR4 polymorphism Asp299Gly and compared response characteristics with similar cells from patients (four) with the wild-type TLR4 genes. Cytokine responses and transcriptome profiles of gingival epithelial cell primary culture cells to TNFα challenge were similar for all primary epithelial cell cultures. P. gingivalis challenge, however, gave markedly different responses for Asp299Gly heterozygous and wild-type epithelial cell cultures. The epithelial cells heterozygous for the TLR4 polymorphism Asp299Gly were functionally hypo-responsive, evidenced by differences in BD-2 mRNA expression, mRNA response profile by microarray analysis and by pro-inflammatory and chemokine cytokines at the protein and mRNA level. These findings emphasize variance in human epithelial cell TLRs, linked with Asp299Gly carriage, which results in a hypo-responsive epithelial cell phenotype less susceptible to Gram-negative diseases and associated systemic conditions.

Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid†

BACKGROUNDnFetal Alcohol Syndrome (FAS), a severe consequence of the Fetal Alcohol Spectrum Disorders, is associated with craniofacial defects, mental retardation, and stunted growth. Previous studies in C57BL/6J and C57BL/6N mice provide evidence that alcohol-induced pathogenesis follows early changes in gene expression within specific molecular pathways in the embryonic headfold. Whereas the former (B6J) pregnancies carry a high-risk for dysmorphogenesis following maternal exposure to 2.9 g/kg alcohol (two injections spaced 4.0 h apart on gestation day 8), the latter (B6N) pregnancies carry a low-risk for malformations. The present study used this murine model to screen amniotic fluid for biomarkers that could potentially discriminate between FAS-positive and FAS-negative pregnancies.nnnMETHODSnB6J and B6N litters were treated with alcohol (exposed) or saline (control) on day 8 of gestation. Amniotic fluid aspirated on day 17 (n = 6 replicate litters per group) was subjected to trypsin digestion for analysis by matrix-assisted laser desorption-time of flight mass spectrometry with the aid of denoising algorithms, statistical testing, and classification methods.nnnRESULTSnWe identified several peaks in the proteomics screen that were reduced consistently and specifically in exposed B6J litters. Preliminary characterization by liquid chromatography tandem mass spectrometry and multidimensional protein identification mapped the reduced peaks to alpha fetoprotein (AFP). The predictive strength of AFP deficiency as a biomarker for FAS-positive litters was confirmed by area under the receiver operating characteristic curve.nnnCONCLUSIONSn: These findings in genetically susceptible mice support clinical observations in maternal serum that implicate a decrease in AFP levels following prenatal alcohol damage.

Genome-wide transcriptome expression in the liver of a mouse model of high carbohydrate diet-induced liver steatosis and its significance for the disease

PurposeTo perform a large-scale gene profiling of the liver in a mouse model of fatty liver induced by high carbohydrate (sucrose) diet (HCD) to gain a deeper insight into potential mechanisms of diet-induced hepatic steatosis.MethodsC57BL/6 male mice were fed either a purified, control diet or a HCD for 16xa0weeks. HCD feeding led to marked liver steatosis without inflammation or necrosis. The expression of 42,500 genes/sequences was assessed.ResultsA number of genes (471) underwent significant expression changes in HCD- as compared to standard diet-fed mice (nxa0=xa05/group; Pxa0<xa00.01). Of these genes, 211 were down- and 260 up-regulated. The latter group includes 20 genes encoding enzymes involved in carbohydrate conversion to fat. The genes that underwent expression changes perform a large variety of molecular functions, and the vast majority of these have never been tested before in non-alcoholic fatty liver of nutritional origin. They reveal novel aspects of the disease and allow identification of candidate genes that may underlie the initiation of hepatic steatosis and progression to non-alcoholic steatohepatitis.ConclusionsHCD-fed laboratory animals provide a model of early non-alcoholic fatty liver disease resembling the disease in humans. The genome wide gene profiling of the liver reveals the complexity of the disease, unravels novel aspects of HCD-induced hepatic steatosis, and helps elucidate its nature and mechanisms.